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Objective: Vascular endothelial growth factor (VEGF) contributes to diabetic retinopathy, but control of its expression is not well understood. Here, we tested the hypothesis that hyperglycemia mediates induction of VEGF expression in a eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) 1 and 2 dependent manner.Research Design and Methods: The retina was harvested from control and type 1 diabetic rats and mice and analyzed for VEGF mRNA and protein expression as well as biomarkers of translational control mechanisms. Similar analyses were performed in Müller cell cultures exposed to hyperglycemic conditions. The effect of 4E-BP1 and 4E-BP2 gene deletion on VEGF expression was examined in mice and in mouse embryo fibroblasts (MEFs).Results: Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes. Increases in expression of 4E-BP1 protein mirrored those of VEGF and expression of 4E-BP1 mRNA was unchanged. Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions. Importantly, the diabetes-induced increase in VEGF expression was not observed in mice deficient in 4E-BP1 and 4E-BP2, nor in MEFs lacking the two proteins.Conclusions: Hyperglycemia induces VEGF expression through cap-independent mRNA translation mediated by increased expression of 4E-BP1. Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites. These findings provide new insights into potential targets for treatment of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF)-induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability. [ABSTRACT FROM AUTHOR]

Objective: Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated.Research Design and Methods: Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization.Results: IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas.Conclusions: These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy is characterized by early onset of neuronal cell death. We previously showed that insulin mediates a prosurvival pathway in retinal neurons and that normal retina expresses a highly active basal insulin receptor/Akt signaling pathway that is stable throughout feeding and fasting. Using the streptozotocin-induced diabetic rat model, we tested the hypothesis that diabetes diminishes basal retinal insulin receptor signaling concomitantly with increased diabetes-induced retinal apoptosis. The expression, phosphorylation status, and/or kinase activity of the insulin receptor and downstream signaling proteins were investigated in retinas of age-matched control, diabetic, and insulin-treated diabetic rats. Four weeks of diabetes reduced basal insulin receptor kinase, insulin receptor substrate (IRS)-1/2-associated phosphatidylinositol 3-kinase, and Akt kinase activity without altering insulin receptor or IRS-1/2 expression or tyrosine phosphorylation. After 12 weeks of diabetes, constitutive insulin receptor autophosphorylation and IRS-2 expression were reduced, without changes in p42/p44 mitogen-activated protein kinase or IRS-1. Sustained systemic insulin treatment of diabetic rats prevented loss of insulin receptor and Akt kinase activity, and acute intravitreal insulin administration restored insulin receptor kinase activity. Insulin treatment restored insulin receptor-beta autophosphorylation in rat retinas maintained ex vivo, demonstrating functional receptors and suggesting loss of ligand as a cause for reduced retinal insulin receptor/Akt pathway activity. These results demonstrate that diabetes progressively impairs the constitutive retinal insulin receptor signaling pathway through Akt and suggests that loss of this survival pathway may contribute to the initial stages of diabetic retinopathy. [ABSTRACT FROM AUTHOR]